Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice. A common variation is to physically sort particles based on their properties, so as to purify populations of interest.
Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.
Flow cytometry can thus be used to analyse objects and separate objects from others in a liquid sample. Hereby objects are suspended in a liquid and are lined up one-by-one, typically by using a low-density suspension of objects and drop-shaped nozzle out of which the object liquid is allowed to fall downwards in a narrow stream or in small droplets. The narrow stream passes in front of illumination means, typically a laser beam or a fluorescence activation light. The laser beam can scatter from the objects, or the illumination means can induce fluorescence in the objects if these have been marked with fluorescent markers. Based on the observed scattered and/or fluorescent response light, information about the individually scanned object can be obtained. This information can furthermore be used to separate this object from the beam. Hereto, a charge can be induced on the object or the liquid droplet in which the object is suspended, and the object can then be removed from the stream by applying an electrostatic field. Important in existing flow cytometry is that the microscopic particles such as objects are lined up one-by-one in a liquid flow when they pass through a measuring apparatus as the measuring apparatus is typically capable of obtaining information about the objects one at a time.
U.S. Pat. No. 7,463,366 discloses a method and device for obtaining a sample with three-dimensional microscopy, in particular a thick biological sample and the fluorescence field emitted by the sample. One embodiment includes obtaining interferometric signals of a specimen, obtaining fluorescence signals emanating from the specimen, recording these signals, and processing these signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time. Another embodiment includes a digital holography microscope, a fluorescence excitation source illuminating a specimen, where the microscope and the fluorescence excitation source cooperate to obtain interferometric signals of the specimen and obtain fluorescence signals emanating from the specimen, means for recording the interferometric signals and fluorescence signals, and means for processing the interferometric signals and the fluorescence signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time.
Patent application WO2004102111 discloses a compact microscope able to work in digital holography for obtaining high quality 3D images of samples, including fluorescent samples and relatively thick samples such as biological samples, said microscope comprising illumination means at least partially spatially coherent for illuminating a sample to be studied and a differential interferometer for generating interfering beams from said sample on the sensor of an electronic imaging device, said interferometer comprising namely tilting means for tilting by a defined angle one the interfering beams relatively to the other, said tilting resulting into a defined shift of said interfering beam on the sensor of the electronic imaging device, said shift being smaller than spatial coherence width of each beam, said microscope being able to be quasi totally preadjusted independently from the samples so that minimum additional adjustments are required for obtaining reliable 3D images of samples.
Hydrodynamic focusing is a technique used by e.g. microbiologists to provide more accurate results from flow cytometers or Coulter counters for e.g. determining the size of bacteria or cells. Cells are counted as they are forced to pass through a small tunnel, causing disruptions in a laser light beam or electricity flow. These disruptions are analyzed by the instruments. It is hard to create tunnels narrow enough for this purpose using ordinary manufacturing processes, as the diameter must be in the magnitude of micrometers, and the length of the tunnel should exceed several millimeters. Hydrodynamic focusing solves this problem by building up the walls of the tunnel from fluid, using the effects of fluid dynamics. A wide (hundreds of micrometers in diameter) tube made of glass or plastic is used, through which a “wall” of fluid called the sheath flow is pumped. The sample is injected into the middle of the sheath flow. If the two fluids differ enough in their velocity or density, they do not mix: they form a two-layer stable flow. The stability is required for a better quality of the measurement of the suspended objects.
WO 2011/068764 discloses a flow cytometer which includes a capillary having a sample channel, at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the sample channel to acoustically concentrate particles flowing within a fluid sample stream in the sample channel; and an interrogation source having a violet laser and a blue laser, the violet and blue lasers being configured to interact with at least some of the acoustically concentrated particles to produce an output signal. A system as in WO 2011/068764 is an example of a flow cytometer with acoustic focusing, and more specifically a capillary-flow cytometer with acoustic focusing.
WO 1998/057152 discloses a method and apparatus for detecting a fluorescent substance tagged to a microparticle are described. The device comprises a single capillary flow carrier system for transporting the microparticle past a selected location, a source of electromagnetic radiation for irradiating the substance tagged to the microparticle, and a detection system for measuring fluorescent light emitted from the substance at the selected location. The method comprises transporting the microparticle to a selected location, irradiating a fluorescent substance tagged to the microparticle, and measuring the fluorescent light emitted from the fluorescent substance at the selected location. A system as in WO 1998/057152 or in WO 2011/068764 is an example of a capillary-flow cytometer.
Prior art flow cytometers have a number of disadvantages. One disadvantage is that the information which is obtained about the object from scattered light is limited. Since the resulting data from flow cytometric analysis is at an aggregate level, it is not easy to observe and measure individual object behavior. Another major disadvantage with a prior art flow cytometer is its low object throughput rate. Even for high-speed flow cytometers and sorters, this is still less than a few thousand objects per second. The throughput rate is related to the flow speed and is limited by the measuring apparatus, which needs to be able to provide a measurement of adequate quality on a moving particle. Typically, the quality of the measurement decreases when the flow speed increases and vice versa. A faster measuring technique, i.e. a technique doing measurements of adequate quality in a smaller period, thus allows for a higher throughput rates. Many experiments require a very large number of objects. This implies that even high-speed flow cytometers and sorters as available in the prior art need to run for long durations, which is not only an expensive proposition but may also pose quality issues because the objects sorted from such long runs may no longer be usable in scientific experiments. This problem may be further aggravated when the sorted objects need to be sterile.
Although high speed flow cytometers can give sterile objects, this makes the operation complex and further reduces the throughput. Most existing flow cytometric systems do not obtain an image of the objects, which may be desired, e.g. for later inspection or for updating an image database. In systems that do obtain an image of the objects, including biological organisms such as cells, bacteria, yeasts, micro-organisms, nematodes and non-biological objects, impurities, contaminants, or any combination thereof, the image is taken with a classical, e.g. a fluorescence or a projection, microscope, which necessitates lining up the objects in the focus of the microscope. Measurement results from objects which are lined up out of focus are usually rejected, which leads to a significant loss in efficiency and in information.
Further, flow cytometry has very sophisticated instrumentation, whereby only skilled and highly trained operators can run it and get any acceptable levels of performance from such an apparatus.
Also, flow cytometers are expensive instruments to purchase and maintain. A laser flow cytometer, which can only analyze but not sort, can be very expensive, especially for small laboratoria, while arc-lamp-based cytometers are only marginally cheaper. Flow cytometers with the additional sorting capability can cost almost double their cheaper versions. Additionally, operating a high-speed sort is another recurring expense that typically costs a substantial amount for each run.
The data acquired with the analysis or measuring apparatus of a prior art flow cytometer may not be accurate enough, it may not be obtained quickly enough, the apparatus may be too expensive, it may only give two-dimensional and/or analogue images. Prior art flow cytometers may use classical optical techniques to obtain an image of the object, whereby the object needs to be in the focus of the optical system. This has two main disadvantages: (i) one may not be able to obtain a good image from an object as it may not be straightforward to place the object in-focus, especially if it is a moving object as in a flow cytometer, and (ii) one can only obtain an image of one object at a time as only one object can be placed in-focus at a time. Furthermore, the gathered sample may need to be processed before analysis, which can be a time-consuming and labor-intensive procedure. Contamination may be an issue when the same apparatus is used to monitor or analyze different samples.
DHMs may provide images and/or directly digitalized information about samples which are superior to images and information obtained by other imaging or analysis techniques. Using a DHM as measurement apparatus of a flow cytometric system or in addition to a flow cytometer allows a user to obtain a three-dimensional picture of the objects suspended in the liquid. This picture is based on the recorded interferometric information recorded by the DHM. A DHM may obtain this information without the necessity of lining up the objects one-by-one in the focus of the microscope. In fact, the interferometric pattern recorded by the DHM allows post-acquisition focusing, resulting in the possibility of extracting a clear 3D image of an object from the digitalized interferometric pattern, preferably by post-acquisition software on an analysis computer, and this without the need for positioning the objects in the focus of a microscope or adapting the focus of the microscope to the position of the object. Moreover, the objects do not need to be lined up single file as a recorded interferometric pattern may comprise 3D information of a large number of objects. This leads to higher throughput rates when a DHM is used in or in conjunction with a flow cytometric system. Furthermore, the DHM may still be used in conjunction with fluorescent dyes or markers, the combination of which leads to an extensive set of observable parameters of a large number of suspended objects and quantities in a single run.
There remains a need in the art for a flow cytometer with improved measuring, observation, analysis and/or separation properties. Using a digital holographic microscope as a measurement apparatus of a flow cytometer will allow to obtain better information about the scanned objects than prior art flow cytometers, especially due to its post-acquisition focusing capabilities which eliminate the need for a focusing apparatus or focusing step in the imaging process. A DHM is furthermore well adapted to provide high-quality information about moving objects as it needs little time to make an interferometric or holographic image of an object. A holographic image contains substantially more information about an object than prior art measurements in flow cytometric systems, which are typically based on scattered and fluorescent light, can deliver.
There remains a need in the art for flow cytometers with an improved throughput rate. The quick acquisition times of a DHM allows a high throughput rate, higher than prior art flow cytometers, especially prior art cytometers which provide a similar amount and quality of information about the scanned objects. The DHM is also capable or gathering information of many objects in one recording step, especially due to its post-acquisition focusing capabilities. Hereby, it is not necessary to present the objects one-by-one in the focus of the microscope, but a hologram of many objects may be obtained.
There remains a need in the art for flow cytometers which are non-invasive to the objects which are scanned. This includes flow cytometers which need to introduce fluorescent or other markers into the objects, including biological organisms such as cells, bacteria, yeasts, micro-organisms, nematodes and non-biological objects, impurities, contaminants, or any combination thereof, which are to be scanned. Such marking can be expensive, time-consuming and invasive to the objects, whereby the object may not be used in further experiments anymore. Using a flow cytometer with a DHM solves this problem as a DHM provides a non-invasive and cost- and time-effective way of obtaining high-quality information about objects. No dyes or markers are needed for the same or an improved quality of the measurements. However, there is also a need in the art for flow cytometers which are capable of obtaining the combined information about suspended objects, as obtainable by a DHM and by fluorescence techniques, in a single run.
There further remains a need in the art for flow cytometers which are easily operated. A DHM can be made easy to operate and furthermore lends itself perfectly for automatisation as it offers digitalized information which can be stored electronically and/or easily transferred for further use.
There also remains a need in the art for flow cytometers which are cheaper to manufacture and to operate. Flow cytometers with a DHM have no need for dyes or markers, which results in lower operation costs. Furthermore, DHMs may comprise a partially coherent light source such as a LED, OLED, OLET or similar, instead of a highly-coherent light source such as a laser, hereby drastically reducing manufacturing costs.
The invention therefore aims to provide a flow cytometer comprising a digital holographic microscope for the observation of the objects in a liquid flow.
DHMs may provide images and/or directly digitalized information about samples which is superior to other imaging or analysis techniques. A DHM does not need a focusing system, as one can perform post-acquisition focusing techniques on the recorded interferometric pattern. Therefore, one can obtain a high-quality image of an object without the need of focusing, and one can obtain images of more than one object at a time, as these images can be acquired in a post-acquisition step. Furthermore, using prior art techniques, the gathered sample may need to be processed before analysis, which can be a time-consuming and labor-intensive procedure. Contamination may be an issue when the same apparatus is used to monitor or analyze different reactors, or the same reactor at different positions of times. Prior art techniques may not always provide the possibility of returning the sample to the reactor or to another reactor, or the possibility of real-time monitoring and providing timely feedback for adapting the reactor's environmental parameters.